The pathophysiology of inflammatory bowel disease (IBD), and Clostridium difficile toxin-associated colitis, two common GI clinical entities with significant morbidity and mortality, is not completely understood. Recent evidence indicate that binding of neuropeptides to receptors on intestinal epithelial cells plays a critical role in the pathogenesis of colonic inflammation. We showed that neurotensin (NT), a neuropeptide released in the colon in response to several stimuli, triggers NF-kappaB activation and release of proinflammatory cytokines from the colonic mucosa. Moreover, high affinity NT receptors are expressed on colonic epithelial cells and are upregulated in IBD and C. difficile toxin-mediated enterocolitis. However, the molecular and biochemical mechanism(s) by which NT binding triggers these responses are not known. Our hypothesis is that the peptide NT, acting through G -protein coupled type 1 receptors (NTR1) on colonic epithelial cells, stimulates signal transduction pathways involving PKC, MAPK and NF-kappaB leading to IL-8 release. Aim 1 will examine the role of the NF- kappaB/IkappaB system in IL-8 gene expression following neurotensin stimulation in colonic epithelial cells. Experiments in this aim will elucidate the role of MAP kinase pathways in NTR1-induced NF-kappaB activation and IL-8 gene expression and examine the importance of PKC-epidermal growth factor communication in MAP kinase activation following NTR1 stimulation. Aim 2 will determine the functional role of the Rho family of small GTP binding proteins RhoA, Rac, and Cdc42 in NT- induced IL-8 expression and determine whether NT binding to NTR1 activates the Rho family proteins. Experiments in this aim will also identify which heterotrimeric G protein receptor subtype is involved in NTR1-induced NF-kappaB activation and IL-8 gene expression, as well as activation of Rho family proteins. Studies described in aim 3 will identify the structural determinants of NTR1 that mediate NT-induced IL-8 expression. Specifically, we will examine the role of the third intracellular loop and the C-terminus of NTR1 in NT signaling and IL-8 expression in human colonocytes. Studies are also proposed to identify the critical residues within the third intracellular loop and the C-terminus involved in NTR1-mediated IL-8 production. Our results will provide important insights on the role of NT and its high affinity receptor in colonic inflammation and may lead to novel therapeutic approaches for the treatment of intestinal inflammation.